Hemochromatosis is a hereditary disorder in which the excessive accumulation of iron in organs such as the liver, heart, and pancreas leads to their damage and dysfunction. Mutations in repulsive guidance molecule c (RGMc) / hemojuvelin (HJV) cause juvenile hemochromatosis, an aggravated form of this disorder that presents earlier in life. Patients with juvenile hemochromatosis, and RGMc knockout mice, have diminished expression of the key iron-regulatory peptide, hepcidin, suggesting that RGMc plays a critical role in the regulation of iron homeostasis through hepcidin;however the mechanisms of RGMc actions are unknown. Recent studies have shown that RGMc binds to the growth factor, bone morphogenetic protein 2 (BMP2), and it has been postulated that this interaction regulates iron metabolism by controlling hepcidin production. The focus of this project is to define the structural determinants of RGMc that mediate its interaction with BMP2, with the long-range goal of understanding the role of RGMc in iron regulation and its dysregulation in disease. The major hypothesis to be tested is that RGMc mutations perturb protein structure such that binding to BMP2 is disrupted. To reach this goal, the follwing specific aims are proposed: I. To define the secondary and tertiary structure of RGMc. The secondary structure of soluble RGMc isoforms will be elucidated via CD measurements. Through a combination of enzymatic digests under reducing and non-reducing conditions, reverse-phase chromatography and mass spectrometry the disulfide bonding pattern of soluble RGMc isoforms will be established. Analogous studies performed on disease-associated amino acid substitution RGMc mutants will determine if point mutations perturb the structure of the protein. II. To characterize the nature of the interaction of RGMc with BMP2. Through competition binding assays the affinity of BMP2 for RGMc will be determined. Further binding studies combined with 2D analysis will determine if phosphorylated RGMc preferentially binds BMP2. By use of disease-associated frameshift mutations in solution- and solid-phase binding assays the binding domain of RGMc for BMP2 will be established. To determine if RGMc modulates BMP function, early events in BMP2 signaling will be monitored upon treatment with soluble RGMc isoforms and mutants. Relevance to public health: These studies will help to better understand the mechanisms of iron regulation, and identify what abnormalities lead to excessive iron accumulation in disease.